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J Virol, July 1998, p. 6244-6246, Vol. 72, No. 7
The Center for Blood Research and Harvard
Medical School Department of Pathology, Boston, Massachusetts
Received 10 November 1997/Accepted 9 April 1998
Fragments of intercellular adhesion molecule 1 (ICAM- 1)
containing only the two most N terminal of its five immunoglobulin SF
domains bind to rhinovirus 3 with the same affinity and kinetics as a
fragment with the entire extracellular domain. The fully active
two-domain fragments contain 5 or 14 more residues than a previously
described fragment that is only partially active. Comparison of X-ray
crystal structures show differences at the bottom of domain 2. Four
different glycoforms of ICAM- 1 bind with identical kinetics.
Intercellular adhesion molecule 1 (ICAM- 1, CD54) is a cytokine-inducible cell surface receptor that
binds to leukocyte integrins LFA-1 and Mac-1 (21).
ICAM- 1 is also the receptor for the major group of human
rhinoviruses (9, 25, 26) and Plasmodium falciparum-infected erythrocytes (2, 18). ICAM- 1
contains five immunoglobulin superfamily (IgSF) domains and is heavily glycosylated. The binding site for rhinovirus is at the tip of domain 1 in the flexible BC and FG loops and extends about halfway down domain 1 (1, 7, 17, 20, 23). The binding site for LFA-1 is on the
edge of domain 1, centered on the C and D strands (1, 7, 8,
24). Soluble ICAM- 1 (sICAM) inhibits infection by rhinovirus,
both by acting as a competitive inhibitor and by irreversible
disruption of the capsid with release of the viral RNA (5, 10, 14,
15). However, ICAM- 1 truncated after what was predicted
(24) to be the last residue of domain 2, F185, showed
markedly reduced inhibition by both mechanisms compared to ICAM- 1
containing all five IgSF domains (10, 15). ICAM- 1 is
unusually heavily glycosylated, and glycosylation is important for
solubility (16). There are conflicting reports on the
importance of the presence or absence of glycosylation for binding to
rhinovirus (13, 16, 17); the influence of different classes
of N-glycans, i.e., glycoforms, has not been explored. The kinetics,
equilibria, and thermodynamics of IC1-5D binding to rhinovirus have
been measured by using surface plasmon resonance (4, 6). The
on-rate constant (kass) for binding of IC1-5D to
rhinovirus is much slower than for binding to an antibody. This is
consistent with either binding to a relatively inaccessible site in the
rhinovirus canyon (19) or a requirement for a conformational
change in the virus to permit binding. The kass
is biphasic, and two putative classes of binding sites have Kds of about 0.7 and 10 µM (6).
Here, we determined the kinetics with truncation and glycosylation
variants of ICAM- 1.
ICAM- 1 truncated after residue 185 (IC1-2D/185), 190 (IC1- 2D/190), 199 (IC1-2D/199), 268 (IC1-3D), or 452 (IC1-5D) was
expressed in insect SF9 cells (IC1-5D/SF9, IC1-3D, and IC1-2D/185),
wild-type CHO-K1 cells (IC1-5D/wt), or lectin-resistant CHO Lec 3.2.8.1 cells (IC1-5D/Lec, IC1-2D/199, and IC1-2D/190) (4, 6, 7, 14). Proteins were purified with monoclonal antibody (MAb)
R6.5-Sepharose and size exclusion chromatography (6).
ICAM- 1 containing only one carbohydrate residue per N-linked site
(IC1-5D/D) was from the flowthrough from a concanavalin A-Sepharose
column that was loaded with IC1-5D/CHOLec treated with 4 mU of
endoglycosidase H (endo H)/µg for 4 h at 37°C in 0.1 M
phosphate buffer (pH 5.5). Purified human rhinovirus 3 (HRV3)
(4) or rabbit anti-mouse Fc IgG (Pharmacia) was linked via
amino groups to the dextran surface of BIAcore sensor chips to a
density of about 10,000 or 8,000 resonance units, respectively
(4). Sensorgrams were subjected to linear transformation to
obtain kinetic constants (11). Binding of
[3H]leucine-labeled HRV3 to HeLa cells and assay of HRV3
disruption by sucrose gradient centrifugation were done as previously
described (15).
IC1-5D/wt was resistant to endo H and sensitive to neuraminidase,
whereas IC1-5D/CHOLec and IC1-5D/SF9 were fully susceptible or
only partially susceptible to endo H, respectively (Fig.
1). Thus, IC1-5D/wt and IC1-5D/Lec
have complex-type and high-mannose N-linked glycans,
respectively. Partial resistance of IC1-5D/SF9 to endo H may
reflect fucosylation (3), which is absent
in CHO Lec 3.2.8.1 cell N glycans (22). IC1-5D/D has
only one N-acetylglycosamine per N-linked site.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Domain Structure of ICAM-1 and the Kinetics
of Binding to Rhinovirus
ABSTRACT
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FIG. 1.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of sICAM- 1 glycosylation variants treated
with endoglycosidases. IC1-5D obtained from wild-type CHO-K1 (CHOwt),
lectin-resistant CHO (CHOLec), or SF9 insect cells were treated in the
presence or absence of endo H, neuraminidase (Neura.), or
N-glycanase (N-Glyc.), whereas the preparation of endo
H-deglycosylated material, IC1-5D/D (D), was run without further
treatment. Positions of molecular weight markers are shown on the left
(molecular weights are in thousands).
The four different IC1-5D glycoforms exhibited no significant differences in the kinetics of binding to HRV3 (Table 1). Association was biphasic, possibly representing two different classes of binding sites (6). A single dissociation rate constant (kdiss) was seen (6).
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Compared to IC1-5D and IC1-3D, three different batches of IC1-2D/185 associated with HRV3 with the same kinetics but reproducibly dissociated five- to sixfold faster (Table 2). This agrees with lower activity in inhibition of binding and in inactivation of rhinovirus (10, 15). By contrast, IC1-2D/199 reproducibly reacted with HRV3 with kinetics indistinguishable from those of IC1-5D and IC1-3D (Table 2). At a later stage in this work, we also tested the IC1-2D/190 fragment. Although less data are available, it clearly was at least as active as IC1-5D, IC1-3D, and IC1-2D/199 (Table 2).
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The association kinetic constant (kass) for binding of ICAM- 1 fragments to MAbs was 10- to 100-fold faster than that for binding to HRV3 (Table 3). The kass increased as the number of Ig-like domains in sICAM- 1 decreased; this is likely to be related to faster diffusion of the shorter molecules, particularly in the dextran matrix to which the MAbs and virus are linked in BIAcore. The lack of such an effect on kass for HRV3 is consistent with the finding that this kass is much slower and, thus, not diffusion limited. Three MAbs to domain 1 of ICAM- 1 (12), LB2 (Table 3), 7F7, and MAY.029 (not shown), and one MAb to domain 2, R6.5 (Table 3), dissociated from ICAM- 1 fragments with kinetics that were unaffected by the truncation position. By contrast, with MAb RR1/1 to domain 1 of ICAM- 1, the IC1-2D/185 fragment dissociated 11.0-fold ± 0.8-fold more rapidly from IC1-2D/185 than from IC1-2D/190, IC1-2D/199, IC1-3D, or IC1-5D. These results confirm the differences between IC1-2D/185 on the one hand and IC1-2D/190 and IC1-2D/199 on the other.
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The crystal structures have recently been determined for two
independent molecules of IC1-2D/190 (7) and one molecule of mutIC1-2D/185 that is identical to IC1-2D/185 except that it contains three Asn
Gln mutations that eliminate N-linked glycosylation sites
(1). The three structures are quite similar overall, but the
position of truncation has a marked effect on the structure of the
bottom of domain 2. The backbone hydrogen bonds between F185 in the G
strand and residues 98 and 100 in the A' strand are lost in the
185-residue fragment, and there are significant shifts in the C
positions of residues 183 and 184 in the G strand, 95 to 97 in the A'
strand and in the connector between the A and A' strands on the edge of
domain 2, 150 to 153 in the EF loop, and 101 and 102 in the A'B loop.
Most significantly, Val186 appears to form an important component of
the bottom of domain 2, with its hydrophobic side chain packing onto
the side chain of Val100 and the hydrophobic portion of the Arg150 side
chain. In the structure of the 185-residue fragment, Val186 is missing,
the side chain of Phe185 is rotated 180°C, and the bottom of domain 2 is less compact overall. The A'G beta-sheet ladders at the bottom of
domains 1 and 2 are structurally homologous in IC1-2D/190, and the
Tyr83 and Trp84 side chains occupy orientations similar to those of Phe185 and Val186. Thus, the structure of Phe185 and Val186 in IC1-2D/190 is appropriate for a domain 2-3 connection structurally homologous to the domain 1-2 connection.
Our data show that structural changes at the bottom and side of domain
2 can alter the affinity of rhinovirus interaction with the top portion
of domain 1 and with MAb RR1/1 to domain 1. Although this may seem
surprising, the conformation of domain 1 has previously been shown to
be influenced by mutagenesis of domain 2, including a conservative
Ala178 Gly mutation in the bulge of strand G near the bottom of
domain 2 (23). In the three structures captured in crystals,
there appear to be no differences in domain 1 that are significant for
rhinovirus binding; however, the structure in solution may differ from
that in crystals. We suggest that in solution, IC1-2D/185 exists in an
equilibrium between two conformations, i.e., (i) an ordered
conformation that can bind rhinovirus and corresponds to the
conformation that crystallizes and (ii) a conformation in which a
portion of domains 1 and 2 is disordered and in which the molecule
cannot bind or binds markedly less well to rhinovirus and MAb RR1/1.
The IC1-2D/190 fragment has a different conformation at the bottom of
domain 2 and appears to be present in an ordered conformation in
solution a much higher proportion of the time than the
185-residue fragment. Equilibration between the ordered and
disordered forms of the IC1-2D/185 fragment that is fast compared to
the rates of binding to and dissociation from HRV3 and antibodies would
be consistent with our kinetic data. Our findings emphasize the
intricacy and delicacy of protein structure and have important
implications for viral receptors and the design of viral antagonists.
Furthermore, our results show that domains 1 and 2 of ICAM- 1 are
sufficient for high-affinity binding to HRV3.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant AI31921.
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FOOTNOTES |
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* Corresponding author. Mailing address: The Center for Blood Research and Harvard Medical School Department of Pathology, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 278-3200. Fax: (617) 278-3232. E-mail: springer{at}sprsgi.med.harvard.edu.
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J Virol, July 1998, p. 6244-6246, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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